The overall goal is to fill substantial gaps in our existing knowledge about the medically important Pyruvate Dehydrogenase Multi-Enzyme Complex (PDHc), and provide the most detailed information possible regarding its' structure-function relationships. PDHc plays a critical role in cellular energy production and carbohydrate metabolism, with disruptions or malfunctions in it associated with numerous pathologies. Bacterial PDHc was also recently recognized as the target for an antibiotic, TPBC, currently in hospital use that is highly effective against emerging health threats including MRSA (methicillin resistant staphylococcus aureus) and VRE (vancomycin resistant enterococci), but its inhibition mechanism is unknown. We will focus on obtaining details for the overall assembly and unusual catalytic process employed by PDHc's, and in particular, for the bacterial PDHc from E. coli. We will also examine differences between the E. coli and human versions. The overall reaction, converting pyruvate to acetyl-CoA while releasing NADH, is carried out by at least three different enzymes with multiple copies of each in large complexes having different architectures. The long-term goal is to understand structure-function relationships in the intact, octahedral, PDHc complex from E. coli and compare them with the icosahedral PDHc complex from humans. We will determine and analyze specific interactions required for governing the assembly of, and catalysis within, the multi-enzyme complex. Unlike most biochemical pathways operating by simple diffusion of substrates between isolated enzymes, PDHc employs complex architectures utilizing substrate channeling involving long and flexible swinging-arms to ?hand-deliver? intermediates between the multiple (at least 60) E1, E2, and E3 enzymatic component active sites. We successfully completed structural analyses of the E1 and E3 components, the E2 core, and some key sub-complexes. This work forms the basis of the current proposal to obtain details for some key missing interactions, and combine them with our earlier data to assess the entire assembly. We will determine and analyze crystal structures for the few missing binary complexes, and correlate the results with overall biological function. We will also provide structure/function information on protein-protein interactions governing assembly/catalysis in the human, icosahedral, PDHc complex, which differs significantly from the octahedral E. coli version. In addition, we will structurally determine/analyze human PDHc components and their binary complexes to assess if differences relative to E. coli PDHc may be exploitable for anti-bacterial drug development, and to determine the structural basis for diseases associated with single residue mutations in human PDHc proteins. We will then put everything together, by analyzing the entire E. coli PDHc structure either by cryo-EM or x-ray methods. Structural studies on isolated proteins or protein-protein complexes will be coupled with biochemical data to obtain complete pictures of the processes. Achieving the aims is the next step in understanding the overall structure and behavior of the entire ~4.6 x 106 Dalton, E. coli PDHc complex.